Ptp1b

Manufactured by Santa Cruz Biotechnology

Sourced in United States

PTP1B is a protein tyrosine phosphatase enzyme that plays a role in the regulation of cellular signaling pathways. It functions by dephosphorylating target proteins, thereby modulating their activity. The core function of PTP1B is to catalyze the removal of phosphate groups from specific tyrosine residues on target proteins.

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5 protocols using ptp1b

Western Blot Analysis of Insulin Signaling

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After determining the total protein content, the Laemmli was added to the supernatant buffer (21 (link)) containing 100 mM DTT and heated in boiling water for 5–10 minutes. Next, equal amounts of protein from the gastrocnemius (50 µg) were used for application in polyacrylamide gel and separation by electrophoresis. The membranes were incubated with: antibodies against: phospho-Y972 IR (#GTX25678) from GeneTex^®; phospho-Y612 IRS1 (#44816G) from Life Technologies^®; Akt (#4685), phospho-Ser473 Akt (#9271), phospho-Ser9 GSK3β (#5558), GSK3β (#5676), phospho-Ser632/635 IRS1 (#2388), RhoA (#2117), PDK (#3062), phospho-Ser241 PDK (#3061), PTEN (#9188), phospho-Ser380 PTEN (#9151), and GAPDH (#2118) from Cell Signaling Techonology^®; phospho-T696 MYPT1 (#92590) from Millipore^®; ROCK2 (#5561), PTP1B (#14021) from Santa Cruz Biotechnology^®; RhoE form Sigma^®. Antibodies were diluted 1:1000. After overnight incubation with primary antibody, the membranes were washed with TBS+Tween 0.05% for 30 min, incubated with horseradish peroxidase secondary antibodies (1:2000 dilution; Cell Signaling Techonology^®) for 1 h and washed again with TBS+Tween 0.05% for 30 min. The bands were visualized with enhanced chemiluminescence and quantified by an Image J program (v1.52a, NIH).

Muñoz V.R., Gaspar R.C., Severino M.B., Macêdo A.P., Simabuco F.M., Ropelle E.R., Cintra D.E., da Silva A.S., Kim Y.B, & Pauli J.R. (2021). Exercise Counterbalances Rho/ROCK2 Signaling Impairment in the Skeletal Muscle and Ameliorates Insulin Sensitivity in Obese Mice. Frontiers in Immunology, 12, 702025.

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Molecular Mechanisms of Metabolic Dysregulation

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Cholesterol, HDL cholesterol, and triglyceride concentrations were determined using kits purchased from GTLab (Buenos Aires, Argentina). Antibodies for eIF2α, p-eIF2α (Ser51), ERK, p-ERK (Thr202/Tyr204), JNK, p-JNK (Thr183/Tyr185), NOX2, NOX4, p47phox, β-tubulin, MCP-1, TNFα, p-PERK (Thr980), PERK, PTP1B, sXBP1, cATF6, IRE1α, p-IR (Tyr1162/Tyr1163), and IR were from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies for p-PKCδ (Thr505), p65, p-p65, IKβα, p-IκBα (Ser32), IKKα, p-IKKα/β (Ser178/180), p-AKT (Ser473), AKT, and were obtained from Cell Signaling Technology (Danvers, MA). Antibody for p-IRE1α (Ser724) was purchased from Abcam (Cambridge, MA). Antibodies for p-IRS1 (Tyr608) and IRS1 were from Millipore Corp. (Billerica, MA). Catalogue numbers for all antibodies are included in supplemental table 1. PVDF membranes and protein standards were obtained from BIO-RAD (Hercules, CA). The ECL Western blotting system was from Thermo Fisher Scientific Inc. (Piscataway, NJ). Fructose was purchased from Saporiti Labs (Buenos Aires, Argentina). EC and all other reagents were from the highest quality available and were purchased from Sigma (St. Louis, MO).

Bettaieb A., Vazquez Prieto M.A., Rodriguez Lanzi C., Miatello R.M., Haj F.G., Fraga C.G, & Oteiza P.I. (2014). (-)-Epicatechin mitigates high fructose-associated insulin resistance by modulating redox signaling and endoplasmic reticulum stress. Free radical biology & medicine, 72, 247-256.

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Tris DBA Cytotoxicity Assay Protocol

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Tris DBA, MTT, Tris, glycine, NaCl, SDS, BSA, IL-6, EGF, and pervanadate were procured from Sigma-Aldrich. The structure of Tris DBA is provided in Figure 1A. The stock solution of Tris DBA (10 mmol/L stock) was prepared in dimethylsulfoxide (DMSO) and stored at 4 °C. Tris DBA was further diluted with a cell culture medium as per the requirements. Cell culture media (Dulbecco’s Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute (RPMI)), and fetal bovine serum (FBS) were purchased from Life Technologies. Rabbit or mouse monoclonal and polyclonal antibodies against Bak, Bcl-2, PARP, survivin, Mcl-1, SHP-2, and PTP1B were obtained from Santa Cruz Biotechnology. Antibodies against phospho-STAT3 (Tyr705), STAT3, phospho-JAK1/2, JAK1/2, caspase-3, phospho-Src, Src, cyclin D1, SHP1, and β-Actin were purchased from Cell Signaling Technology. The BioRender program was used to generate the graphical abstract of this paper.

Arora L., Mohan C.D., Yang M.H., Rangappa S., Deivasigamani A., Kumar A.P., Kunnumakkara A.B., Garg M., Chinnathambi A., Alharbi S.A., Alahmadi T.A., Rangappa K.S., Hui K.M., Sethi G, & Ahn K.S. (2021). Tris(dibenzylideneacetone)dipalladium(0) (Tris DBA) Abrogates Tumor Progression in Hepatocellular Carcinoma and Multiple Myeloma Preclinical Models by Regulating the STAT3 Signaling Pathway. Cancers, 13(21), 5479.

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Synthesis and Identification of LXQ46

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LXQ46 (3,4-dibromo-5-(5-(4-(4-ethoxyphenoxy)phenyl)oxazol-2-yl)benzene-1,2-diol) was synthesized and identified by our lab (purity 98%). Primary antibodies used in this study: PTP1B (#133259) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PARP (#9532), Bcl-xL (#2764), Phospho-AMPK (Thr172, #2535), AMPK (#2793S), Phospho-Akt (Ser473, #4060), Akt (#4685), Phospho-ERK (Thr202/Tyr204, #4370S), ERK (#4696S), Phospho-PRAS40 (#2640) and Ki67 (#9027) were purchased from Cell Signaling Technology (Danvers, MA, USA). Bax (#32503), Bcl-2 (#32124), CDK2 (#32147), CDK4 (#108357), CDK6 (#124821), Cyclin D1(#134175), Phospho-Src (Tyr529, #32078), Src (#47405), Phospho-PI3K (#182651), PI3K (#86714), PRAS40 (#151719) were purchased from Abcam (Cambridge, MA, USA). Phospho-p70 S6 Kinase (Thr389/412, #3228) and p70 S6 Kinase (#6226), Phospho-PKM2 (Tyr105 #7771), PKM2 (#5234) were purchased from Affinity Biosciences (Cincinnati, OH, USA). β-actin antibody and all secondary antibodies were obtained from Proteintech Group (Wuhan, China).

Xu Q., Wu N., Li X., Guo C., Li C., Jiang B., Wang H, & Shi D. (2019). Inhibition of PTP1B blocks pancreatic cancer progression by targeting the PKM2/AMPK/mTOC1 pathway. Cell Death & Disease, 10(12), 874.

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Regulation of Caveolin-1 and β-Catenin

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All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise. NS1643 was from Alamo Laboratories Inc. (San Antonio, TX, USA). Calpeptin was purchased from Tocris Bioscience (Minneapolis, MN, USA). PTP1B inhibitor was from Cayman Chemical (Ann Arbor, MI, USA). Phospho-Y14, total Caveolin-1, β-catenin, and caspase-3 antibodies were from BD Biosciences (San Jose, CA, USA). Phospho-Y416, Y527, and total Src were from Cell Signaling Technology (Danvers, MA, USA). PTP1B, desmoplakin, desmoglein, and plakophilin monoclonal Abs, normal mouse IgG, and protein A/G agarose beads were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). DAPI and all fluorescently labeled secondary antibodies were purchased from Molecular Probes (ThermoFisher Scientific, Waltham, MA, USA). Active β-catenin monoclonal antibody was purchased from MilliporeSigma (Burlington, MA, USA). HRP-conjugated goat-anti-mouse and goat-anti-rabbit secondary antibodies were from KPL (Gaithersburg, MD, USA). Lipofectamine 2000, ECL Super Signal kit, and Crosslink magnetic IP/Co-IP kit were from ThermoFisher Scientific (Waltham, MA, USA).

Jiang Y., Senyuk V., Ma K., Chen H., Qin X., Li S., Liu Y., Gentile S, & Minshall R.D. (2022). Pharmacological Activation of Potassium Channel Kv11.1 with NS1643 Attenuates Triple Negative Breast Cancer Cell Migration by Promoting the Dephosphorylation of Caveolin-1. Cells, 11(15), 2461.

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